Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain
Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. were originally isolated based on their ability to bind the double-stranded (dsDNA) sequence 5-CCAAT-3 (29). Later on work defined the Y-box element as 5-CTGATTGG(C/T)(C/T)AA-3, which contains a reverse CCAAT package (in boldface). This regulatory element is found in the promoter regions of many vertebrate gamete-specific genes, including the oocyte-specific gene, the rat testis-specific histone gene, and the murine testis-specific gene (37). Subsequent work has shown that some Y-box family members possess specificity for binding both pyrimidine-rich dsDNA and single-stranded DNA (16). These observations have led to several models for Y-box protein function including DNA interactions, such as functions in transcriptional rules, Jujuboside A chromatin changes, and DNA restoration. Many Y-box proteins have also been identified as components of messenger ribonucleoprotein particles (mRNPs). Y-box protein p50 is the major core protein of cytoplasmic mRNPs of somatic cells in rabbits (10). p50 and the poly(A) binding protein are the two most abundant proteins in these mRNPs. In oocytes, Y-box proteins will also be abundant components of mRNP3+4s comprising masked mRNAs (26). Proteins homologous to mRNP3+4s are indicated during murine spermatogenesis and form complexes with stored mRNAs (21). Murine Y-box protein MSY1 has also been shown to be associated with germ cell mRNPs during spermiogenesis (34). MSY2 and MSY4 are components of a 48- and 50-kDa RNA binding activity present in murine testis components (8). This activity is definitely a component of testis mRNPs comprising protamine mRNAs. The protamines are small arginine-rich proteins involved in condensation of DNA in the nuclei of adult spermatids. The protamine mRNAs are synthesized in round and early-elongating spermatids, transported to the cytoplasm, and stored as translationally repressed mRNPs until their translation from 2 to 8 days later on in elongated spermatids (2, 20). The MSY2 and MSY4 proteins bind a 22-nucleotide (nt) region of the 3 untranslated region (UTR) and a 20-nt region of the 3 UTR (11). The 22-nt region lies within the 1st 37 nt of the 3 UTR and may delay the translation of an hGH transgene in vivo (12). MSY2 is the murine orthologue of protein FRGY2 (mRNP3+4) and was cloned from an expression library display with anti-FRGY2 antibodies (15). FRGY2 was originally cloned by its ability to bind the CCAAT element (35), but as mentioned above is also found associated with germ collection mRNPs. FRGY2 is consequently considered to have a dual function: a role in transcriptional activation of oogenic genes and a second part in masking gametogenic mRNAs. was cloned from a mouse testis cDNA library using the candida three-hybrid system with the first 37 nt of the protamine 1 (mRNA storage. In this study, we have delineated a site present in the 3 UTR of the 3 UTR that murine Y-box proteins MSY2 and MSY4 bind specifically. Single-nucleotide mutations within the conserved Y-box acknowledgement site (YRS) get rid of binding of MSY2 and MSY4 in vitro and in the candida three-hybrid system. Furthermore, transgenic experiments with mice suggest that the YRS will also function in vivo. MATERIALS AND METHODS Mice. C57BL/6J male mice were purchased from Jackson Laboratory (Pub Harbor, Maine) and were sacrificed by carbon dioxide asphyxiation. Transgenic mice were generated by microinjecting a purified DNA fragment at a concentration of 2 ng/l in 10 mM Tris (pH 7.5)C0.25 mM EDTA into pronuclei of fertilized eggs derived from FVB/N FVB/N Jujuboside A (Taconic Labs, Germantown, N.Y.) matings (6). Pseudopregnant B6 CBA F1/ (Taconic Labs) foster females were utilized for oviduct implantation of eggs that survived microinjection. Transgenic animals were recognized by PCR. Three lines of mice (3505, 3514, and 3515) were generated and Jujuboside A analyzed. The expression of each transgenic collection was analyzed by Northern analysis. Line 3505 indicated the KRT17 transgene at a lower level.